1. Field of the Invention
The present invention relates to the use of cyclic dinucleotides to attenuate virulence of microbial pathogens and to inhibit biofilm formation, thereby controlling microbial colonization or infections caused by a wide variety of microbial species.
2. Description of the Related Art
Cholera is an important diarrheal disease of humans that results in significant morbidity and mortality (Pollitzer, 1959; and Kaper et al., 1995). Cholera affects more than 75 countries and every continent (Communicable Disease Surveillance and Response, World Health Organization, who.org). Cholera is acquired by drinking fecally contaminated food or water containing pathogenic Vibrio cholerae that can colonize the small intestine and release cholera toxin (CT) resulting in massive secretory diarrhea and death if untreated (Kaper et al., 1995). Because of its high death-to-case ratio, persistence in water supplies and its ability to occur in explosive epidemic form, cholera is a public health concern. Furthermore, because of the potential threat of weaponized V. cholerae to the food and water supply, it is a priority organism in biodefense research. The threat to the economy, environment and human health is also highlighted by the finding that V. cholerae has the potential to be transported internationally and invade new regions through the ballast water of ships (McCarthy et al., 1994). V. cholerae is known to persist in the environment, however, the factors promoting the environmental persistence of V. cholerae are not well understood.
V. cholerae can alter its phenotype and reversibly switch from EPSoff (smooth colony morphology) to EPSon (rugose colony morphology) in which the cells are embedded in extracellular polysaccharide or rugose exopolysaccharide (rEPS) and display a wrinkled “rugose” colony morphology (FIGS. 1A and 1B) and an associated biofilm (White, 1940 and Rice et al., 1993). The switch to EPSon and the rugose phenotype promotes biofilm formation (Rice et al., 1993; Morris et al., 1996; and Watnick et al., 1999). Importantly, EPS is essential for V. cholerae biofilm formation. The rugose variant is highly chlorine resistant and shows increased resistance to killing by acid, UV light and complement-mediated serum bactericidal activity (Rice et al., 1993; Morris et al., 1996; and Yildiz et al., 1999). Therefore, switching to EPSon and the rugose phenotype might be important in niche specialization and in promoting survival and fitness in particular environments. Rugose strains are virulent and cause fluid accumulation in rabbit ileal loops, produce diarrhea in human volunteers and are highly resistant to complement-mediated bactericidal activity (Rice et al., 1993; Morris et al., 1996; and Yildiz et al., 1999). The rugose or wrinkled colony phenotype consisting of aggregating cells has been reported in S. enterica Enteritidis (Petter, 1993), S. enterica Typhimunium (Anriany et al., 2001), V. parahaemolyticus (Güvener et al., 2003), P. aeruginosa (Parsek, 2003), and Enterobacter sakazakii (Farmer et al., 1980). Research by the laboratory of the present inventor and others has also shown that production of V. cholerae EPS is linked to the type II general extracellular protein secretion pathway which is also involved in secretion of important virulence factors (Ali et al., 2000; Davis et al., 2000).
The vps (Vibrio polysaccharide) gene cluster in V. cholerae carries the structural genes for the biosynthesis of rEPS (Yildiz et al., 1999). The vps gene cluster is thought to be comprised of two closely located but separate operons in which vpsA and vpsL represent the first genes of each operon (Yildiz et al., 1999 and 2001). Transcription of vpsA and vpsL is regulated by VpsR (a homolog of σ54 transcriptional activators) by a mechanism that is not well understood (Yildiz et al., 2001). VpsR has high homology to NtrC, AlgB and HydG bacterial enhancer-binding protein that activates transcription after phosphorylation of its receiver domain by an associated sensor kinase protein (Kern et al., 1999). Previous studies have found that HapR in some V. cholerae strains is linked to the rugose phenotype by some unknown mechanism (Jobling et al., 1997) and CytR can repress transcription of vps genes and the associated biofilm formation (Haugo et al., 2002). The present inventor has also found that switching to the rugose phenotype in V. cholerae is independent of ToxT, LuxS and RpoS (Ali et al., 2002). However, the molecular basis underlying switch from the smooth to the rugose phenotype of V. cholerae is still not well understood.
Early studies on the rugose phenotype of V. cholerae were impeded by the very low frequency of switching to EPSon and the rugose phenotype under the conditions tested (Morris et al., 1996; Yildiz et al., 1999; and Wai et al., 1998). The laboratory of the present inventor recently identified conditions that promote the rapid shift (up to ˜80%) to the rugose phenotype in a process called high frequency rugose production (HFRP) (Ali et al., 2002). It was found that there are differences in the expression and stability of the phenotype between epidemic strains and that the ability to switch at high frequency was more common in epidemic V. cholerae strains than in nonpathogenic strains (Ali et al., 2002). This suggests that the ability to switch to the rugose phenotype is important in V. cholerae and might provide an adaptive advantage under specific conditions.
Biofilms are the primary mode of existence of many bacterial species and are central to their survival, persistence and often virulence (Costerton et al., 1995; Davey et al., 2000; Donlan, 2002 and Watnick et al., 2000). Biofilms resist environmental stresses and adverse conditions better than free-living cells, have increased nutrient availability and can better avoid immune responses (Anwar et al., 1992). A common feature of biofilms is that microorganisms are embedded in an extracellular matrix comprised mostly of EPS (Costerton et al., 1981 and Wingender et al., 1999). EPS is important for the structural and functional integrity of biofilms and determines its physicochemical and biological properties and has a role in adhesion, protection and facilitates community interactions (Wimpenny, 2000). EPS provides protection from a variety of environmental stresses such as UV radiation, pH shifts, osmotic shock, and desiccation.
The role of biofilms in the environmental persistence and transmission of certain pathogens is also well recognized. Like V. cholerae (Ali et al., 2002; Morris et al., 1996 and Yildiz et al., 1999), Salmonella enterica Typhimurium has the ability to form a rugose EPS-producing phenotype which has increased biofilm forming ability and is proposed to have a role in increased persistence in the environment (Anriany et al., 2001). Salmonella enteritidis biofilms resistant to cleaning fluids have been shown to persist for at least 4 weeks in domestic toilets after episodes of salmonellosis (Barker et al., 2000). The finding that E. coli and Salmonella biofilms can be found on sprouts may make their eradication with antimicrobial compounds difficult and therefore increasing their persistence, resulting in ingestion and infection (Fett, 2000). The importance of biofilms is also highlighted in the process of horizontal gene transfer since some results suggest that DNA exchange may be increased in bacteria that are attached to a surface and in biofilms rather than between free-swimming planktonic cells (Ehlers, 2000). This has implications in the transfer of genes encoding functions such as antibiotic resistance or virulence and overall persistence.
Clinically, biofilm formation is known to be a key factor in the establishment and persistence of several difficult to treat infections. Cystic fibrosis is caused by certain P. aeruginosa strains which express copious amounts of EPS and form biofilms in the lung (Davies et al., 1995; Geesey et al., 1993 and Govan et al., 1996). The EPS of these P. aeruginosa strains makes them recalcitrant to antimicrobial treatment.
Interestingly, like the EPS of V. cholerae (Ali et al., 2002 and Morris et al., 1996), alginate EPS production by P. aeruginosa protects these strains against chlorine and may contribute to survival of these bacteria in chlorinated water systems (Grobe et al., 2001). Another example of a biofilm-mediated infection is chronic ear infection (otitis media) (Dingman et al., 1998). Peridontitis is also another example of a biofilm-mediated disease that results from chronic inflammation of the tissue supporting the gums and can lead to tooth loss. The main microbe causing this disease is Porphyromonas gingivalis (Lamont et al., 1998).
The EPS matrix of biofilms has the potential to physically prevent access of certain antimicrobial agents into the biofilm by acting as an ion exchanger, thereby restricting diffusion of compounds from the external milieu into the biofilm (Goodell et al., 1985; Nichols et al., 1988 and Nickel et al., 1985). Helicobacter pylori produces a biofilm that appears to be important in enhancing resistance to host defense factors and antibiotics and in promoting growth under low pH conditions in vivo (Stark et al., 1999). Biofilm bacteria can be up to 1,000-fold more resistant to antibiotic treatment than the same organism grown planktonically (Gilbert et al., 1997). Clinical biofilm infections are marked by symptoms that typically recur even after repeated treatments with antibiotics. Moreover, biofilm infections are rarely resolved by the host's immune system (Costerton et al., 1999). Bacterial biofilms on prosthetic valves are the leading cause of endocarditis in patients who have undergone heart valve replacement. Among patients who develop these infections, the mortality rate is as high as 70% (Hyde et al., 1998). Millions of catheters (e.g., central line, intravenous, and urinary catheters) are inserted into patients every year, and these implants serve as a potential surface for biofilms. Overall, it is thought that upwards of 60% of all nosocomial infections are due to biofilms. These biofilm-based infections can increase hospital stays by up to 2-3 days and cost upwards of $1 billion per year in added costs (Archibald et al., 1997).
Staphylococcus aureus is another biofilm-forming bacteria that has long been recognized as an important human and animal pathogen (Archer, 1998; Hermans et al., 2003; Kluytmans et al., 1997 and Sutra et al., 1994). S. aureus can be found on the skin and mucosal surfaces of humans, particularly the anterior nares. If followed over time, ˜20% of the human population are persistent carriers; ˜60%, intermittent carriers while ˜20% of the population will never be colonized (Peacock et al., 2001). S. aureus is a common cause of both community-acquired and hospital-acquired infections. In a recent population-based active surveillance study from Canada, the annual incidence of invasive S. aureus infection was 28.4 per 100,000 population (Laupland et al., 2003). Certain populations including patients with indwelling medical devices such as vascular catheters, patients on hemodialysis, patients who use intravenous drugs, patients with dermatologic disease and diabetes mellitus have higher rates of colonization than the general population (Kirmani et al., 1978; Tuazon et al., 1975 and 1974). The S. aureus carrier state is clinically important because a carrier is at risk for infection with the colonizing strain. Studies in patients on dialysis, patients with HIV infection and patients with bloodstream infection support the hypothesis that S. aureus isolates causing infection are endogenous in origin when strains are examined by molecular typing (Ena et al., 1994; Luzar et al., 1990; Nguyen et al., 1999; von Eiff et al., 2001 and Yu et al., 1986). Hence, ways to inhibit or reduce S. aureus carriage and colonization are needed.
According to the Center for Disease Control and Prevention's National Nosocomial Infection Surveillance system, S. aureus is particularly a common cause of nosocomial infections and is the most common cause of surgical site infection and the second most common cause of nosocomial bacteremia (National Nosocomial Infections Surveillance (NNIS) Report, 1998). The overall number of S. aureus infections in intensive care units increased from 1987 to 1997 with the majority of the increase due to S. aureus isolates resistant to methicillin (Lowry, 1998). S. aureus is often resistant to multiple antibiotics. Infections caused by methicillin- and multiple antibiotic resistant S. aureus (MRSA) are particularly difficult to treat and MRSA infections are often associated with higher mortality and increased healthcare costs than methicillin-sensitive strains (Cosgrove et al., 2003).
S. aureus is also a common cause of intramammary infections (IMI) in lactating females and often results in chronic mastitis with annual losses in the dairy industry associated with subclinical mastitis in dairy cows across the U.S. being estimated at approximately $1 billion (Ott, 1999). The drug of choice for infections due to methicillin-resistant S. aureus (MRSA) is vancomycin, although this antibiotic is given as a last line of treatment.
Like other bacterial species, biofilm formation is known to be a key factor in the establishment and persistence of staphylococcal infections. Bacterial cells in biofilms can be up to 1,000-fold more resistant to antibiotic treatment than the same cells grown planktonically. Consistent with this observation, biofilm formation on tissues or on medical devices is an important first step in the pathogenesis of S. aureus infection of humans and animals (Bradley et al., 1991; Cole et al., 2001; Cucarella et al., 2001, 2002 and 2004; Götz, 2002; Huang et al., 2003; Kluytmans et al., 1997; Mest et al., 1994; Muder et al., 1991; Peacock et al., 2001; Pujol et al., 1996; and Roghmann et al., 2001). Overall, it is thought that upwards of 60% of all nosocomial infections involve biofilms. These biofilm-based infections can increase hospital stays by up to 2-3 days and cost upwards of $1 billion per year in added costs. Although the risk of infection is high in people colonized with S. aureus, there is another compelling reason to prevent colonization and biofilm formation, which is to prevent the transmission of S. aureus to others (Muto et al., 2003). MRSA does not spontaneously emerge from existing methicillin-susceptible S. aureus. The majority of people with MRSA colonization acquire MRSA through exposure to the hands of healthcare workers transiently colonized with MRSA from prior contact with an MRSA infected or colonized patient (Muto et al., 2003). Infection control measures such as isolation and handwashing reduce but do not eliminate this transmission, although compliance with these policies is often low (Richet et al., 2003). Decolonization regimens, as an approach to controlling transmission to others, have generally been unsuccessful as the eradication of MRSA is generally only temporary. Therefore, the development of novel intervention strategies that prevent or inhibit colonization and biofilm formation are needed.
Cyclic nucleotides, such as cAMP and CGMP, are well recognized as important low-molecular weight signaling molecules in eukaryotes. In bacteria, while cAMP has a role in alleviating glucose catabolite repression (Jackson et al., 2002; Notley-McRobb et al., 1997), cGMP has not been shown to act as a signaling molecule. However, another guanosine nucleotide, the cyclic dinucleotide c-di-GMP (also known as 3′,5′-cyclic diguanylic acid, cyclic diguanylate, cyclic diguanosine monophosphate, cyclic bis(3′→5′)diguanylic acid, cyclic diguanylic acid, cGpGp, and c-GpGp)
where G in the above structure is guanine, has been reported to be an intracellular bacterial signaling molecule in a few species and whose structure is known and consists of two cGMP molecules bound head-to-tail (Jenal, 2004 and Ross et al., 1991). c-di-GMP was first identified in Acetobacter xylinum (renamed Gluconacetobacter xylinum) and shown to regulate cellulose production in this species (Amikam et al., 1989; Mayer et al., 1991; Ross et al., 1990 and 1991). The exact molecular mechanism remains unclear but regulation in G. xylinum appears to involve c-di-GMP binding to a membrane protein that activates gene expression. Cellulose production appears to be modulated by the opposing effects of two proteins with GGDEF domains, diguanylate cyclase (Dgc) and c-di-GMP phosphodiesterase (PdeA), each controlling the level of c-di-GMP in the cell. Thus, c-di-GMP is thought to be a signaling molecule.
Based on studies by the laboratory of the present inventor and others, it is now becoming increasingly reported that biofilm formation in many pathogens including Vibrio cholerae, Yersinia pestis, Salmonella enteritidis Typhimurium and Pseudomonas aeruginosa is associated with GGDEF proteins (Bomchil et al., 2003; D'Argenio et al., 2002; Jones et al., 1999 and Römling et al., 2000).
The increasing emergence of antimicrobial resistance in bacterial pathogens and the importance of colonization and biofilm in the infection process requires that alternate antimicrobial strategies be developed. Until the present invention, the application of cyclic dinucleotides such as c-di-GMP for use as an antimicrobial approach in the control of biofilms and potentially infection has not been described.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.